A Pilot Study of CK19, CK20 and GCC mRNA in the Peripheral Blood as a Colorectal Cancer Biomarker Panel.

Colorectal cancer remains one of the major cancer- related deaths despite progress in the treatment during past decades. Detection of disease at earlier stages reduces its mortality. The aim of current study was to investigate expression of Cytokeratin 19 (CK19), Cytokeratin 20 (CK20) and Guanylyl Cyclase C (GCC) mRNA in peripheral blood of non- metastatic colorectal cancer patients which may result into introducing of an early detection test. 25 patients with colorectal cancer and 25 healthy controls were recruited. Blood was obtained from all individuals. Expression of CK19 and CK20 and GCC mRNA and 18SrRNA (as reference gene) were determined based on real- time RT-PCR on total RNA from blood. CK19, CK20 and GCC expression had been detected in 68%, 76% & 52% of patient group, respectively, which was higher than healthy group, with 8%, 32% and 0% expression, respectively (p<0.05). CK20 was over-expressed 8- fold more in patients compared to controls. Similar result was found for CK19 with 4- fold over- expression. Sensitivity and specificity of combination of markers were 88% and 68%, respectively. Current data suggest that the detection of CK20 & CK19 as relative sensitive markers may become a valuable tool for primary diagnosis of colorectal cancer in early stages. GCC could be considered as a specific tumor marker for detection of colorectal cancer. Higher expression of these markers in patients may be considered as a relative good tool for the diagnosis of disease in non- metastatic stages.

espite significant progress in decreasing mortality and improving survival rates in patients, cancer is still the major cause of death worldwide (1). Colorectal cancer is the third most commonly diagnosed cancer in males and the second in females (2). During the last decades, considerable progress has been achieved towards improving survival of patients. Since more than 60% of colorectal cancer has been identified at the symptomatic phases with lower rate of long-term survival, diagnosis of disease at the earlier asymptomatic stages and effective screening is and prognostic biomarkers will enable oncologists to individualize therapeutic strategies by increasing drug effectiveness and decreasing incompatible side effects in colorectal cancer patients (4,5).
The use of Real-time-PCR to analyze the blood of cancer patients for the detection of mRNA expressed in tumor cells is one of the interesting concepts in the management of cancer (6). Many mRNA markers of colorectal cancer have been studied in the peripheral blood of the patients (7). in peripheral blood (9,10). Cytokeratin is one of the intermediate filaments, which is mainly found in epithelial cells and particularly useful tools in oncology diagnostics. Cytokeratin-based tumor marker assays may be considered as simple, noninvasive, cheap, and reliable predictive tests and offer a tool for more efficient management before conventional methods (11).
Cytokeratin 20 (CK20) is a low molecular weight member of the Cytokeratin family of proteins that is expressed in primary colorectal tumors and their metastases (12). Cytokeratin19 (CK19), because of restricted range of its expression, has been considered as a tool for detecting and identifying cancer cells in the peripheral blood by PCR analysis. It has previously been shown to serve as circulating tumor cells associated marker in colorectal cancer patients and has been used to detect disseminated tumor cells and occult metastases in the patients. So, CK19 could improve the early detection of distant organmetastases (13,14).
Guanylyl cyclase C (GCC), found as the target for heat-stable enterotoxin of Escherichia coli is one of studied biomarkers of colorectal cancer (15).

Study groups
We recruited 25 patients (

Statistical analysis
The sample size had been calculated based on the difference between proportions of positive ratios in two groups, reported in previous similar studies (13,14). All statistics were calculated using the SPSS software (Version 10). T-Test was applied for comparison of two means. Two-sample binomial test was used for comparisons of the prevalence between study groups. A p-value < 0.05 was considered significant. ΔΔCt method was applied for the estimation of difference level of gene expression between studied groups.

Results
The mean age of patient and healthy groups was 61 years (range: 16 -87 years) and 62.3 years (range: 25-88 years), respectively. There was no significant difference between patient and healthy groups in mean of age (P= 0.80). Therefore, this marker could be considered as a reference for the normalization of our biomarkers expression in blood samples.

Expression rate of markers in patients and healthy volunteers
Positive rate of markers in patients and   Comparison of single and marker combined with positive ratios between male and female patients, between colon and rectal cancer patients and between different stages of disease did not show meaningful difference.

Expression levels for the markers between patient and healthy volunteers
To assay the expression levels of markers in patients and healthy groups, we calculated ΔCt of both CK19 and CK20 using the following formula: [Ct value of maker-Ct value of reference] and then, the mean of this value was calculated in two study groups.
For CK19, the mean of ΔCt were 7.73 in patient group and 9.69 in healthy group. These parameters were 7.24 and 10.23 for CK20, respectively. Then, we calculated ΔΔCt of CK19 and CK20 using the following calculation: [ΔCt of patient group -ΔCt of healthy group]. The parameter was -1.96 for CK19 and -2.99 for CK20.
According to Livak method, which explains that 2 -ΔΔCt value should be considered as indicator of difference between the expression level of two groups, expression level of CK19 in patients was approximately calculated 4 (2 1.96 ) times higher than healthy volunteers. For CK20, the expression level was 8 (2 3.99 ) times higher in patient group. None of the subjects in control group showed GCC expression, so we did not include GCC in this part ( Fig.1).

Discussion
The aim of the present pilot study was to find blood biomarkers for the early diagnosis of CRC. In this study, the specificity of GCC was estimated 100%. GCC mRNA expression has been detected in all stages of primary and metastatic CRC and any grade and anatomic location of tumor, but only few studies evaluated peripheral blood GCC expression in CRC patients (25,26).
According to many studies, GCC could be used as a rather specific biomarker for diagnosis, staging of disease and evaluation of treatment efficacy as well as prognosis interpretation of CRC. Sensitivity and specificity of GCC mRNA have been reported in a wide range in different investigations (9,25,26).
Colonoscopy, as gold standard test for CRC diagnosis, has 95% sensitivity and 100% specificity which is higher than the other routine screening procedures of colorectal cancer (27,28). By comparison with colonoscopy, our primary findings showed relatively optimistic results. Therefore, it may be assumed as a tool for detection of nonmetastatic disease in people with no tendency to invasive screening procedures. However, it should not be considered as a replacement method for colonoscopy.
In conclusion, the results of this study suggest that mRNA markers of peripheral blood may be considered as useful tools to find non-metastatic CRC by real-time RT-PCR. Combination of sensitive and specific makers as a panel of diagnostic test and considering cut-off value strategy increase the total sensitivity and specificity of the panel as a primary non-invasive test for the diagnosis of CRC in non-metastatic stages. However, more widespread studies are required to confirm our findings.